Protein digestion



Patented Apr. 14, 1942 Pno'rElN nIGEs'rioN Gilbert n.Y .lm-es, stanford, and Joseph Geom Niedercorn,

Riverside,

Conn., assignors to American Cyanamid Company, New York, N. Y., a corporation of Maine original application June 16, 1939, serial Np.

Divided and this application November 9, 1939, Serial No. 303,556

4 Claims.

The invention relates to the controlled digestion of protein materials and more particularly to the bating of hides and skins by subjecting them to the action of tryptic enzymes of Pencillium brevicaule, otherwise known as Scopulariopsis brevicaulis.

It has been discovered that Penicillium brevicaule produces enzymes whichfhave sumcient proteolytic activity under neutral, acid, and alkaline conditions to render them commercially attractive in the bating of hides and in similar processes involving the selective digestion of proteins.

It is an object of the present invention to provide these enzymes in the form of a dried proteolytic culture which is suitable for use as a bate. It is a further object to provide a method for the hating of hides and skins using these enzymes as bates, preferably in conjunction with a deliming agent such as ammonium sulfate.

The invention includes both the production of these enzymes from Penicillium brevicaule by inoculation of a suiable nutrient medium and cultivation of the inoculated medium and the provision of the enzymate (enzyme-i-carrier) in dried form, and also the selective digestion of proteins and the hating of hides and skins by subjecting the materials to the action of these proteolytic enzymes.

As illustrative of the proteolytic activity in alkaline solution of these enzymes produced by Penicillium brevicaule there is shown in the single iigure of the accompanying drawing a curve, the ordinates of which are given in terms of standard alkali and the abscissae in terms of pI-I values. The curve was constructed from values obtained in digesting a casein substrate (sodium caseinate) with a culture of these enzymes at increasing alkalinities of the sodium caseinate solution. The undigested casein was precipitated with a standardized solution of sulfuric acid and sodium sulfate and the filtrate therefrom, containing the digested casein, titrated with 0.1 N-alkali. The tests were conducted by a modification of the procedure of Volhard and Loehlein for the determination of proteolytic activity of enzymes described in Praktikum der Physiologischen Chemie," Part 1, pages 258-259, by Peter Rona, 2d ed., Julius Springer, Berlin, 1931. The curve which was plotted for a pH range of 7-10, vafter sloping of! from the point of neutrality, changes sign and ascends until a pH of about 9.5 is reached whereupon it again changes sign and slopes downwardly to the pH of 10. By inspection it will be seen from this curve that the enzymes of the present invention manifest a uniformly good rate of activity in alkalies at least up tov the point where the alkalinity reaches a pH value of about 9.5. K

The preparation of these enzymes is asfollows: A suitable nutrient medium in the granular or solid discrete particle condition, such as bran, moistened with an equal weight of water, is inoculated with a culture of Penicillium brevicaule and the inoculated moist bran spread out in thin layers on trays. The inoculated bran is then incubated in an loven maintained at a temperature of about\30 C. and preferably at not higher than this temperature, and at a humidityA in the oven such that the atmosphere therein is saturated but does not contain suicient moisture to cause deposition of the same onto the bran. 'I'he inoculated bran is maintained in the oven until sporulation occurs. After incubation for the optimum period the bran culture may be thoroughly mixed with 0.2% oi cresylic acid in solution, if desired, to improve the wetting out of the branwhen used in solution. The culture or enzymate is then dried at a temperature below 45 C., e. g.,l 40 C., and may be used as such for hating, or an ammonium salt, e. g., ammonium sulfate, may be incorporated with the moist mass and the mixture then dried. The bran used for the culture may or may not be sterilized before the inoculation and likewise the culture may or may not be sterilized, although sterilization of the culture or enzymate does not appea necessary at the present time.

The enzymey may be liberated from the dried culture by elution with dilutesolutions of various salts, such as ammonium chloride, ammonium sulfate, sodium chloride, sodium sulfate, etc. and the eluted enzymes used in other elds as digestants.

The proteolytic enzymes may be applied to the batng of hides and skins in the form of an enzymate in any manner now practiced in the art for the application of other tryptic hates. One method now in use involves washing the hides from the dehairing step, and adding an ammonium salt, such as ammonium sulfate, to the water containing the hides in order to lower their pH which is generally very high due to the strongly alkaline conditions under which dehairing takes place. Before adding the ammonium salt the hides may be treated for removing lime blast by the addition of a suitable acid, such as hydrochloric acid, to the water bath containing the hides. After the pH of the bath has been wesh a soo 1b. pack er nmed yinps nomine .dehairing'bath with water for 15 minutes at '10- F. Heat the washed pack .in'water to 95 F. and add 5 lb.'of hydrochloric acid thereto, the

- bath showing red to methyl orange. After three minutes add lime to the bath until it is slightly` pink to phenolphthalein. To the bath then add on the weight of the kips) of enzymate prepared as described above. Run the paddle for the desired length of bating time with a final temperature therein of 85F. After the kips have been hated-cool them to 70 F.

While the application of these enzymes has beenfdescribed with particular reference to the bating of hides and skins their utilityI is not limited to this field. On the contrary they may be used in many other processes in which a tryptic enzyme of high activity is desired, such as in desizing and dcgumming textiles, paper sizing, tenderizing of meat. stripping of gelatin from photographic illms and plates, manufacture of peptones, chewing gum, glue. foods. drugs and biological products or in any neld where bytheir use a protein or a protein degradation product can be reduced to a lower molecular size of increased solubility.

It will be understood that the above description is intended as illustrative and not as limiting of the invention, the scope of which is de- `lined by the following claims. f

v 'I'his is a division of-our copending application Serial No. 279.472. illed June 16, 1939.

FWhat we claim is:

1. A process of beting hides which comprises subjecting them to the action of enzymes present 10 in a dried culture of the mold Pentcillium brevicaule, said enzymes being characterized by a uniformly high rate of proteolytic activity under alkaline conditions within a pH range up to about 2. A process of bating hides under alkaline conditions within a pH range up to about 9.5 -which comprises subjecting them to the action of enzymes of Pen'tcillium brevicaule in a dried culture of the mold, said enzymes being characterized 3% lb.of ammonium sulfate and about 1% (based n by a uniformly high rate of vproteolytic activity under alkaline conditions within a pH range up to about 9.5.

3. A process of hating hides which comprisessubjecting them to the action of enzymes of Penicillium brevicaule in a dried culture of the alkaline conditions within a pH range up to about l V9.5, and in the presence loi' ammonium sulfate.

GILBERT B. AYRES. JOSEPH G. NIEDERCORN. 

